Enhanced anti-tumor effects by combination of tucatinib and radiation in HER2-overexpressing human cancer cell lines

Abstract

Background: Tucatinib (TUC), a HER2-directed tyrosine kinase inhibitor, is the first targeted drug demonstrating intracranial efficacy and significantly prolonged survival in metastatic HER2-positive breast cancer (BC) patients with brain metastases. Current treatments for brain metastases often include radiotherapy, but little is known about the effects of combination treatment with TUC. Therefore, we examined the combined effects of irradiation and TUC in human HER2-overexpressing BC, non-small cell lung cancer (NSCLC), and colorectal cancer (CRC) cell lines. For the latter two, a standard therapy successfully targeting HER2 is yet to be established.

Methods: Nine HER2-overexpressing (BC: BT474, ZR7530, HCC1954; CRC: LS411N, DLD1, COLO201; NSCLC: DV90, NCI-H1781) and three control cell lines (BC: MCF7, HCC38; NSCLC: NCI-H2030) were examined. WST-1 assay (metabolic activity), BrdU ELISA (proliferation), γH2AX assay (DNA double-strand breaks (DSB), Annexin V assay (apoptosis), and clonogenic assay (clonogenicity) were performed after treatment with TUC and/or irradiation (IR). The relevance of the treatment sequence was analyzed exemplarily.

Results: In BC, combinatorial treatment with TUC and IR significantly decreased metabolic activity, cell proliferation, clonogenicity and enhanced apoptotis compared to IR alone, whereby cell line-specific differences occurred. In the PI3KCA-mutated HCC1954 cell line, addition of alpelisib (ALP) further decreased clonogenicity. TUC delayed the repair of IR-induced DNA damage but did not induce DSB itself. Investigation of treatment sequence indicated a benefit of IR before TUC versus IR after TUC. Also in CRC and NSCLC, the combination led to a stronger inhibition of metabolic activity, proliferation, and clonogenic survival (only in NSCLC) than IR alone, whereby about 10-fold higher concentrations of TUC had to be applied than in BC to induce significant changes.

Conclusion: Our data indicate that combination of TUC and IR could be more effective than single treatment strategies for BC. Thereby, treatment sequence seems to be an important factor. The lower sensitivity to TUC in NSCLC and particularly in CRC (compared to BC) implicates, that tumor promotion there might be less HER2-related. Combination with inhibitors of other driver mutations may aid in overcoming partial TUC resistance. These findings are of high relevance to improve long-time prognosis especially in brain-metastasized situations given the intracranial activity of TUC.

Keywords: Breast cancer; Colorectal cancer; HER2; Non-small cell lung cancer; Radiotherapy; Tucatinib.

Fig. 1

Metabolic activity, proliferation, apoptosis and necrosis of BC cell lines after combined treatment with TUC/IR. Metabolic activity, proliferation, apoptosis and necrosis of BT474, ZR7530, and HCC1954 cells 72 h after treatment with TUC and/or fractionated IR for 4 consecutive days. Data are presented as mean ± SEM of three experiments (n = 3) per cell line, except for ∼ (n = 2). Significant differences relative to the corresponding 0 nM TUC group are indicated by asterisks, between groups marked with a hash symbol. (A) Metabolic activity, measured by WST1 assay, and (B) proliferation, measured by BrdU incorporation assay, are presented relative to untreated non-irradiated control samples (= 1). Experiments were performed in duplicates. (C) Apoptotic (plain) and necrotic (hatched) cell fractions (1 = 100%), measured by Annexin V/PI assay, are shown together with one representative dot plot per cell line. Asterisks and hash symbols indicate significant differences in apoptotic fractions. (D) Phase contrast images of HCC1954 cells without (I) and 72 h after treatment (II, III) with 1 µM TUC and 8 Gy IR (red: PI; scale bar: 100 μm). (E) Metabolic activity, measured by WST1 assay, of HER2-negative cell lines HCC38 and MCF7 (hatched) after treatment with TUC are presented together with HER2-overexpressing cell lines (plain) relative to untreated control samples (= 1)

Fig. 2

Metabolic activity, proliferation, apoptosis and necrosis of CRC and NSCLC cell lines. CRC cell lines (LS411N, DLD-1, COLO201) and NSCLC cell lines (DV90, NCI-H1781, A549) 72 h after treatment with TUC and/or single-dose IR. Data are presented as mean ± SEM of one experiment per cell line (n = 3). Significant differences relative to the corresponding 0 nM TUC group are indicated by asterisks between groups marked with a hash symbol. (A) Metabolic activity, measured by WST1 assay, and (B) proliferation, measured by BrdU incorporation assay, are presented relative to untreated non-irradiated control samples (= 1). Experiments were performed in duplicate (A, NCI-H1781) or triplicate (all others). (C) Apoptotic (plain) and necrotic (hatched) fractions, measured by Annexin V/PI assay, are shown together with one representative dot plot per entity. Asterisks and hash symbols indicate significant differences in apoptotic fractions

Fig. 3

Clonogenic survival of BC, NSCLC and CRC cell lines after combined treatment with TUC/IR. Cells were treated with TUC and irradiated daily for 4 consecutive days. Significant differences to corresponding 0 Gy group are indicated by asterisks. Data are presented as mean ± SEM. (A) Three experiments (n = 3) in triplicate (BT474) or duplicate (HCC1954) are presented as mean ± SEM, except for (∼, n = 2). (B) Joint analysis was conducted for CRC (LS411N, DLD-1: triplicates, n = 1) and NSCLC (DV90, NCI-H1781, A549: triplicates, n = 1) cell lines. (C) Photographs of one colony together with their corresponding Petri dish/well from control as well as maximum treatment group are presented for one cell line per entity (scale bar: 300 μm)

Fig. 4

Detection of DSBs in BC cell lines after combined treatment with TUC/IR. γH2AX staining at 1, 24, 48, and 72 h after treatment of BC cell lines (BT474, HCC1954, ZR7530) with TUC and/or single-dose IR. (A) Number of γH2AX foci/nucleus was counted in 50 cells. Data are presented as mean value ± SEM (n = 1, foci of 50 cells counted). Significant differences relative to corresponding untreated control group are indicated by asterisks, between groups marked with a hash symbol. (B) Fluorescence images of HCC1954 24 h after TUC treatment and/or single-dose IR (blue: DAPI; red: γH2AX foci; scale bar: 50 μm; insert: 4x magnification)

Fig. 5

Effect of TUC/fractionated IR treatment sequence on metabolic activity of BT474 cells. (A) Metabolic activity was measured by WST1 assay. Data from three experiments in duplicate are presented as mean ± SEM (n = 3) relative to untreated control (= 1). Significant differences from the corresponding 0 Gy groups are indicated by asterisks. (B) Treatment schedules

Fig. 6

(A) Metabolic activity of BC, CRC and NSCLC cell lines with different PI3K and HER2 statuses after combined treatment with ALP/TUC. Metabolic activity, measured by WST1 assay, of CRC cell lines DLD1 and COLO201, NSCLC cell line NCI-H2030, and BC cell lines HCC1954 and MCF7 72 h after treatment with ALP and/or TUC. Relative values (untreated control = 1) of a single experiment performed in triplicate are presented as mean ± SEM. Cell lines are marked by shading and color as either HER2-overexpressing (hatched) or HER2-negative (plain) as well as PI3KCA-mutated (red coloring) or PI3KCA-wild type (yellow coloring). (B), (C), (D) Verification of HER2 expression by Immmunofluorescence in cells used for the TUC/ALP combinatorial treatments. (B) Difference in mean fluorescence intensities of HER2 and IgG negative control are presented in five cell lines. Data from two independent experiments are presented as mean ± SEM. (C) Representative histogram overlays of HER2 staining versus IgG negative control for each cell line. (D) Representative photographs of HER2-(Alexa488, green) and nuclear (DAPI, blue) stained floating cells prepared for flow cytometry analysis (scale bar 50 μm)